Apoptotic Programmed Cell Death Research Articles

Virus-induced modulation of apoptotic cell death program

Data of native and foreign authors concerning presence of several adaptive mechanisms in viruses which allow them to influence on key moments of apoptosis process are presented in the paper. Viral destructive action, intensity and efficacy of immune reactions and hence clinical manifestations of infectious process depend on virus-induced modulation of programmed cellular death. Influence of viruses on the program of apoptosis is established to be the basic one in forming viral persistence.

Apoptosis dominant in the periinfarct area of human ischaemic stroke—a possible target of antiapoptotic treatments

Animal experiments have suggested that apoptotic programmed cell death is responsible for an important portion of the delayed ischaemic brain damage. Antiapoptotic signalling through erythropoietin (EPO) binding to its receptor (EPOR) is triggered by systemic or local hypoxia and may exist in the post-ischaemic brain, and a neuroprotective effect by EPO was described recently and proposed for clinical stroke treatment. The objective of the study was to determine whether apoptosis occurs in human ischaemic stroke and to describe its topographical distribution. An autopsy cohort consisting of 13 cases of fatal ischaemic stroke (symptom duration from 15 h to 18 days) treated at the Department of Neurology, Helsinki University Central Hospital and 3 controls were studied. DNA damage was investigated by immunofluorescent TUNEL-labelling in combination with apoptotic cell morphology and by visualization of a major signalling system of apoptosis, Fas-FasL (Fas-ligand), by the immunoperoxidase technique. The relationship of EPO and EPOR in the face of TUNEL-labelled and necrotic cell death was co-registered in human cerebral neurons undergoing different stages of ischaemic change. TUNEL-labelled cells with apoptotic morphology were disproportionately more frequent, 148% (30) [mean (SE)] in the periinfarct versus 97% (22) in the core, as percentage of the cells in the contralateral hemisphere (P = 0.027). The apoptotic cell percentage reached up to 26% (2) of all cells in periinfarct area. A linear correlation was found for Fas and its counterpart FasL expression (r(S) = 0.774, P < 0.001). Ischaemia induced widespread neuronal expression of EPOR, which was inversely related to the severity of ischaemic neuronal necrosis (P < 0.05). To conclude, these data verify the predominance of apoptosis in the periphery of human ischaemic infarctions. Fas and FasL were linearly overexpressed supporting that this 'death-receptor' complex may promote the completion of cell death. Increased EPO signalling may be a cellular response for survival in less severely damaged areas. These results support antiapoptotic therapies against delayed neuronal cell death in human ischaemic stroke.

Hyperosmotic stress induces metacaspase‐ and mitochondria‐dependent apoptosis in Saccharomyces cerevisiae

During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Deltacyc7Delta and cyc3Delta, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.

Cardiolipin oxidation sets cytochrome c free

Cytochrome c release from the mitochondria is a critical component of the apoptotic cell-death program. Cytochrome c–catalyzed peroxidation of cardiolipin, a mitochondrial phospholipid, has now been shown to lessen the binding of cytochrome c to the mitochondrial inner membrane and facilitate permeabilization of the outer membrane. These results describe a new and earlier pro-apoptotic role for cytochrome c.

The apoptosis inhibitory domain of FE65-like protein 1 regulates both apoptotic and caspase-independent programmed cell death mediated by tumor necrosis factor

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.

Glycogen Synthase Kinase 3 Activity Mediates Neuronal Pentraxin 1 Expression and Cell Death Induced by Potassium Deprivation in Cerebellar Granule Cells

Expression of neuronal pentraxin 1 (NP1) is part of the apoptotic cell death program activated in mature cerebellar granule neurons when potassium concentrations drop below depolarizing levels. NP1 is a glycoprotein homologous to the pentraxins of the acute phase immune response, and it is involved in both synaptogenesis and synaptic remodeling. However, how it participates in the process of apoptotic neuronal death remains unclear. We have studied whether the signaling pathways known to control neuronal cell death and survival influence NP1 expression. Both activation of the phosphatidylinositol 3-kinase/Akt (PI-3-K/AKT) pathway by insulin-like growth factor I and pharmacological blockage of the stress activated c-Jun NH(2)-terminal kinase (JNK) offer transitory neuroprotection from the cell death evoked by nondepolarizing concentrations of potassium. However, neither of these neuroprotective treatments prevents the overexpression of NP1 upon potassium depletion, indicating that nondepolarizing conditions activate additional cell death signaling pathways. Inhibiting the phosphorylation of the p38 mitogen-activated protein kinase without modifying JNK, neither diminishes cell death nor inhibits NP1 overexpression in nondepolarizing conditions. In contrast, impairing the activity of glycogen synthase kinase 3 (GSK3) completely blocks NP1 overexpression induced by potassium depletion and provides transient protection against cell death. Moreover, simultaneous pharmacological blockage of both JNK and GSK3 activities provides long-term protection against the cell death evoked by potassium depletion. These results show that both the JNK and GSK3 signaling pathways are the main routes by which potassium deprivation activates apoptotic cell death, and that NP1 overexpression is regulated by GSK3 activity independently of the PI-3-K/AKT or JNK pathway.

Inhibitor of apoptosis proteins: new therapeutic targets in hematological cancer?

Apoptosis is an essential process for the selection and survival of lymphocytes. Resistance to apoptosis can promote malignant transformation of hematopoietic cells. Proteins that regulate apoptosis may therefore be critically involved in the development of hematological cancer. A delicate balance between pro- and antiapoptotic mechanisms determines whether a cell death signal can activate the execution of the apoptotic cell death program. The family of inhibitor of apoptosis (IAP) proteins is a recently identified, novel category of apoptosis-regulatory proteins. IAPs can inhibit the activation of caspases that are the executioners of apoptosis, activated by both the extrinsic and intrinsic pathway. IAPs may thereby set the threshold for apoptosis-activation and play a key role in the regulation of apoptotic cell death. IAPs themselves are also subject to strict regulation through feedback mechanisms. This paper focuses on the role of IAP family proteins in the regulation of apoptosis and discusses implications for their involvement in cancer and possible use for cancer therapy, especially in leukemias and lymphomas.

Noncytotoxic ribonuclease, RNase T1, induces tumor cell death via hemagglutinating virus of Japan envelope vector.

Several ribonucleases, including onconase and alpha-sarcin, are known to be toxic to tumor cells. On the other hand, although its structure is related to that of alpha-sarcin, RNase T1 is noncytotoxic because of its inability to internalize into tumor cells. In this study, we internalized RNase T1 into human tumor cells via a novel gene transfer reagent, hemagglutinating virus of Japan (HVJ) envelope vector, which resulted in cell death. This cytotoxicity was drastically increased by pretreatment of HVJ envelope vector with protamine sulfate, and was stronger than that of onconase, which is in phase III human clinical trials as a nonmutagenic cancer chemotherapeutic agent. Furthermore, internalized RNase T1 induced apoptotic cell death programs. Because its cytotoxicity is unfortunately not specific to tumor cells, it cannot at present be developed as an anticancer drug. However, we believe that RNase T1 incorporated in HVJ envelope vector will be a unique anticancer drug if HVJ envelope vector can be targeted to tumor cells.

Regulation of apoptosis by endoplasmic reticulum pathways.

Apoptotic programmed cell death pathways are activated by a diverse array of cell extrinsic and intrinsic signals, most of which are ultimately coupled to the activation of effector caspases. In many instances, this involves an obligate propagation through mitochondria, causing egress of critical proapoptotic regulators to the cytosol. Central to the regulation of the mitochondrial checkpoint is a complex three-way interplay between members of the BCL-2 family, which are comprised of an antiapoptotic subgroup including BCL-2 itself, and the proapoptotic BAX,BAK and BH3-domain-only subgroups. Constituents of all three of these BCL-2 classes, however, also converge on the endoplasmic reticulum (ER), an organelle whose critical contributions to apoptosis is only now becoming apparent. In addition to propagating death-inducing stress signals itself, the ER also contributes in a fundamental way to Fas-mediated apoptosis and to p53-dependent pathways resulting from DNA damage and oncogene expression. Mobilization of ER calcium stores can initiate the activation of cytoplasmic death pathways as well as sensitize mitochondria to direct proapoptotic stimuli. Additionally, the existence of BCL-2-regulated initiator procaspase activation complexes at the ER membrane has also been described. Here, we review the potential underlying mechanisms involved in these events and discuss pathways for ER-mitochondrial crosstalk pertinent to a number of cell death stimuli.

AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction

Cerebellar Purkinje neurons (PNs) are selectively vulnerable to AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepriopionic acid)-induced delayed neurotoxicity known as dark cell degeneration (DCD) that is expressed as cytoplasmic and nuclear condensation, neuron shrinkage, and failure of physiology. The present study was initiated to determine whether AMPA-receptor-induced DCD in PNs is associated with Bax translocation to the mitochondria, cytochrome C release from the mitochondria, changes in mitochondrial potential, and activation of representative initiator and executor caspases that include caspase-9, caspase-3, and caspase-7. AMPA consistently and rapidly hyperpolarized mitochondria as reflected by an increase in MitoTracker Red CMS Ros fluorescence. Increases in Bax immunoreactivity were quantitatively and temporally variable and Bax failed to localize to mitochondria. Additionally, we observed a marked increase in immunoreactivity of cytochrome C although its release from mitochondria was not apparent. Mitochondrial membrane hyperpolarization and increases in cytochrome C immunoreactivity preceded caspase activation. Immunohistochemical analyses revealed the active form of caspases-3 and -9 were markedly and significantly increased in PNs following 30 μM AMPA, and caspase-9 activation preceded caspase-3. Increases in active caspase-7 immunoreactivity were less frequently encountered in PNs. Thus DCD shares some characteristics of apoptotic programmed cell death, but lacks typical mitochondrial pathophysiology associated with classic apoptosis. These findings suggest that AMPA-induced DCD is a form of active PCD that lies on a spectrum between classical apoptosis and passive necrosis.

Neuroendocrine-immune surveillance of osteosarcoma: emerging hypothesis.

Osteosarcoma is a bone-forming cancer predominantly found in children and adolescents more often than in adults. Osteosarcoma of the gnathic apparatus is relatively rare in the young population, and this condition becomes a concern of clinical dentists for predominantly the middle-aged and aging patient groups. Osteosarcomas are invaded by lymphocytes, which exhibit signs of activation. The immune processes that are engaged within the malignant bone matrix involve the production of cytokines, which regulate the process of apoptotic programmed cell death. This paper discusses the mechanisms by which apoptosis of osteosarcoma cells is modulated by the neuroendocrine-immune system, and potential physiological implications.

Post-ischemic activation of caspase-3 in the rat hippocampus: evidence of an axonal and dendritic localisation

The relationship between caspase-3 activation and delayed neuronal death after ischemia was examined. Expression of caspase-3 was evaluated by colorimetric assay, immunoblotting and by immunohistochemistry. Apoptosis was characterised by terminal desoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labelling. Immunohistochemistry showed caspase-3 activation in the whole hippocampus as early as 30 min after ischemia with exclusive localisation in fiber systems, especially in the perforant path and mossy fibers, Schaffer-collaterals, as well as apical and basal dendrites of pyramidal cells. One day post-ischemia, the 18 kDa cleavage product of caspase-3 (p18) was seen in all cell compartments (nucleus, cytosol and dendrites) throughout the entire subfields and the dentate gyrus with high distribution in mossy fibers. Two days post-ischemia, p18 kDa was only seen in the nuclei and cytosol of hippocampal cells without specific regional differences among hippocampal subfields. A significant number of apoptotic cells appeared only in the CA1 pyramidal cells at 2–3 days post-ischemia. Our data provides the first evidence that caspase-3 activation was detectable in the trisynaptic pathway fiber bundles which probably correspond to perforant path, alvear path and collaterals of Schaffer, and that activation of caspase-3 led to execution of apoptotic cell death program in selectively vulnerable areas, but not in the resistant area of the hippocampus.

Calpain-mediated X-linked Inhibitor of Apoptosis Degradation in Neutrophil Apoptosis and Its Impairment in Chronic Neutrophilic Leukemia

The number of neutrophils in the blood and tissues is controlled by constitutive apoptotic programmed cell death and clearance by phagocytes such as macrophages. Here, we found that calpains cleave the X-linked inhibitor of apoptosis (XIAP) in vitro, producing fragments that are unable to inhibit caspase-3. These fragments were detected in normal neutrophils but were unstable and rapidly degraded. Calpain inhibition delayed tumor necrosis factor-alpha-induced apoptosis of normal neutrophils, consistent with a role for calpains in regulating the onset of apoptosis. Interestingly, neutrophils from three patients with chronic neutrophilic leukemia, a rare syndrome characterized by accumulation of mature neutrophils, exhibited decreased mu-calpain expression, diminished calpain activity, and impaired XIAP degradation. Neutrophils from these patients displayed a delay in spontaneous, Fas-stimulated, and tumor necrosis factor-alpha-induced apoptosis. These observations suggest that calpain-mediated XIAP degradation contributes to initiation of apoptosis in normal neutrophils and dysfunction of this regulatory pathway can lead to pathological neutrophil accumulation.

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.

Invited review: Interplay between molecular chaperones and signaling pathways in survival of heat shock.

Heat shock of mammalian cells causes protein damage and activates a number of signaling pathways. Some of these pathways enhance the ability of cells to survive heat shock, e.g., induction of molecular chaperones [heat shock protein (HSP) HSP72 and HSP27], activation of the protein kinases extracellular signal-regulated kinase and Akt, and phosphorylation of HSP27. On the other hand, heat shock can activate a stress kinase, c-Jun NH2-terminal kinase, thus triggering both apoptotic and nonapoptotic cell death programs. Recent data indicate that kinases activated by heat shock can regulate synthesis and functioning of the molecular chaperones, and these chaperones modulate activity of the cell death and survival pathways. Therefore, the overall balance of the pathways and their interplay determine whether a cell exposed to heat shock will die or survive and become stress tolerant.

Overcoming Resistance to Platinum Therapy in Patients with Advanced Cancer

The platinum-containing compounds cisplatin and carboplatin represent the mainstay of chemotherapeutic treatment for a variety of solid tumors occurring in adults, especially testicular and ovarian cancers. These drugs confer their antitumor efficacy by binding to DNA, especially to guanine bases, either adjacent on the same DNA strand or across strands. Tumour resistance, either present at the onset of therapy or acquired during successive courses of therapy, represents the significant limiting factor to long-term patient survival. From numerous studies of cancer cell lines in vitro, it is apparent that tumor resistance to cisplatin may arise through two general mechanisms: one preventing sufficient platinum from binding to DNA (reduced membrane transport and cytoplasmic inactivation by thiol-containing species such as glutathione or metallothioneins); and a second whereby platinum-DNA damage does not result in cell death (enhanced DNA nucleotide excision repair, increased tolerance, loss of DNA mismatch repair, failure to undergo apoptotic programmed cell death). It remains largely unclear as to the relative importance of these mechanisms in the clinic or whether additional mechanisms also exist.

Apoptosis in yeast: a new model for aging research

Apoptosis is a form of programmed cell death with a central role in development and homeostasis of metazoan organisms. Recent research indicates the presence of an apoptotic cell death program in unicellular eukaryotes. Yeast can be killed by expression of mammalian proapoptotic genes or in response to oxygen stress, which is an inducer of mammalian apoptosis. The dying yeast cells show morphological alterations typical for apoptosis. Yeast provides a simple model for cellular aging. The observation that old yeast cells produce oxygen radicals and die apoptotically may provide clues to a similar sequence of events in mammalian aging.

The autophagosomal-lysosomal compartment in programmed cell death.

In the last decade a tremendous progress has been achieved in understanding the control of apoptosis by survival and death factors as well as the molecular mechanisms of preparation and execution of the cell's suicide. However, accumulating evidence suggests that programmed cell death (PCD) is not confined to apoptosis but that cells use different pathways for active self-destruction as reflected by different morphology: condensation prominent, type I or apoptosis; autophagy prominent, type II; etc. Autophagic PCD appears to be a phylogenetically old phenomenon, it may occur in physiological and disease states. Recently, distinct biochemical and molecular features have been be assigned to this type of PCD. However, autophagic and apoptotic PCD should not be considered as mutually exclusive phenomena. Rather, they appear to reflect a high degree of flexibility in a cell's response to changes of environmental conditions, both physiological or pathological. Furthermore, recent data suggest that diverse or relatively unspecific signals such as photodamage or lysosomotropic agents may be mediated by lysosomal cysteine proteases (cathepsins) to caspases and thus, apoptosis. The present paper reviews morphological, functional and biochemical/molecular data suggesting the participation of the autophagosomal-lysosomal compartment in programmed cell death.

TGF-beta modulates programmed cell death in the retina of the developing chick embryo.

Programmed cell death (PCD) is a key phenomenon in the regulation of cell number in multicellular organisms. We have shown that reduction of endogenous transforming growth factor beta (TGF-beta) prevents apoptotic PCD of neurons in the developing peripheral and central nervous system, suggesting that TGF-beta is an important mediator of ontogenetic neuron death. Previous studies suggested that there are other pro-apoptotic molecules, nerve growth factor (NGF) and brain-derived neurotrophic factor, that induce cell death in the nervous system. In the developing chick retina, NGF induces PCD by activation of the p75 receptor. We have studied the role of TGF-beta and its putative interdependence with NGF-mediated PCD in the chick retina. We found that TGF-beta is present in the developing chick retina during the period of PCD and is essentially required to regulate PCD of retinal cells. TGF-beta 2, TGF-beta 3 and the ligand-binding TGF-beta receptor can be detected immunocytochemically in the central retina, a region where apoptosis is most prominent during the early period of PCD. Application of a TGF-beta-neutralizing antibody to chick embryos in ovo resulted in a decrease in the number of TUNEL-positive cells and a reduction of free nucleosome levels. In terms of magnitude, reduction of PCD caused by the neutralization of endogenous TGF-beta was equivalent to that seen after anti-NGF application. Neutralization of both factors did not result in a further decrease in apoptosis, indicating that NGF and TGF-beta may act on the same cell population. Furthermore, neutralization of TGF-beta did not affect the expression of NGF or the p75-receptor. Our results suggest that TGF-beta and NGF are both required to regulate cell death in the chick retina in vivo.

Elevated vulnerability to oxidative stress-induced cell death and activation of caspase-3 by the Swedish amyloid precursor protein mutation.

The Swedish double mutation (KM670/671NL) of amyloid precursor protein (APPsw) is associated with early-onset familial Alzheimer's disease (FAD) and results in from three- to sixfold increased beta-amyloid production. The goal of the present study was to elucidate the effects of APPsw on mechanisms of apoptotic cell death. Therefore, PC12 cells were stably transfected with human APPsw. Here we report that the vulnerability of APPsw-bearing PC12 cells to undergo apoptotic cell death was significantly enhanced after exposure to hydrogen peroxide compared to human wild-type APP-bearing cells, empty vector-transfected cells, and parent untransfected cells. In addition, we have analyzed the potential influence of several mechanisms that can interfere with the execution of the apoptotic cell death program: the inhibition of cell death by the use of caspase inhibitors and the reduction of oxidative stress by the use of (+/-)-alpha-tocopherol (vitamin E). Interestingly, oxidative stress-induced cell death was significantly attenuated in APPsw PC12 cells by pretreatment with caspase-3 inhibitors but not with caspase-1 inhibitors. In parallel, caspase-3 activity was markedly elevated in APPsw PC12 after stimulation with hydrogen peroxide for 6 hr, whereas caspase-1 activity was unaltered. In addition, oxidative stress-induced cell death could be reduced after pretreatment of APPsw cells with (+/-)-alpha-tocopherol. The protective potency of (+/-)-alpha-tocopherol was even greater than that of caspase-3 inhibitors. Our findings further emphasize the role of mutations in the amyloid precursor protein in apoptotic cell death and may provide the fundamental basis for further efforts to elucidate the underlying processes caused by FAD-related mutations.